Cachexia: A systemic consequence of progressive, unresolved disease.

Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia.

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

Dual-modality gene reporter for in vivo imaging.

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.

MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model.

OBJECTIVES: Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging. DESIGN: The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. (13)C magnetic resonance spectroscopic imaging ((13)C-MRSI) was used to measure non-invasively changes in (13)C labelling of alanine and lactate with disease progression, following injection of hyperpolarised [1-(13)C]pyruvate. RESULTS: Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the [1-(13)C]alanine/[1-(13)C]lactate signal ratio observed in (13)C-MRSI images of the pancreas. CONCLUSIONS: Metabolic imaging with hyperpolarised [1-(13)C]pyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa.

Multiplexing Autoradiography.

Autoradiography, the direct imaging of radioactive distribution in tissue sections, is a powerful technique that has several key advantages for the validation of PET radiotracers. Using autoradiography, we can localize radiotracer uptake to neighbours of cells, and when multiplexed with additional radiotracers, fluorescent probes, or in situ tissue analysis, autoradiography can help to characterize the mechanism of radiotracer uptake and assess functional heterogeneity in tissue. In this chapter, the author outlines the basic ex vivo autoradiography protocol and shows how it can be multiplexed using dual radionuclides 18F and 14C. They also highlight where autoradiography can be combined with other technologies to provide synergistic information for interrogating spatial biology.

Noninvasive Stratification of Colon Cancer by Multiplex PET Imaging.

PURPOSE: The current approach for molecular subtyping of colon cancer relies on gene expression profiling, which is invasive and has limited ability to reveal dynamics and spatial heterogeneity. Molecular imaging techniques, such as PET, present a noninvasive alternative for visualizing biological information from tumors. However, the factors influencing PET imaging phenotype, the suitable PET radiotracers for differentiating tumor subtypes, and the relationship between PET phenotypes and tumor genotype or gene expression-based subtyping remain unknown. EXPERIMENTAL DESIGN: In this study, we conducted 126 PET scans using four different metabolic PET tracers, [18F]fluorodeoxy-D-glucose ([18F]FDG), O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET), 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), and [11C]acetate ([11C]ACE), using a spectrum of five preclinical colon cancer models with varying genetics (BMT, AKPN, AK, AKPT, KPN), at three sites (subcutaneous, orthograft, autochthonous) and at two tumor stages (primary vs. metastatic). RESULTS: The results demonstrate that imaging signatures are influenced by genotype, tumor environment, and stage. PET imaging signatures exhibited significant heterogeneity, with each cancer model displaying distinct radiotracer profiles. Oncogenic Kras and Apc loss showed the most distinctive imaging features, with [18F]FLT and [18F]FET being particularly effective, respectively. The tissue environment notably impacted [18F]FDG uptake, and in a metastatic model, [18F]FET demonstrated higher uptake. CONCLUSIONS: By examining factors contributing to PET-imaging phenotype, this study establishes the feasibility of noninvasive molecular stratification using multiplex radiotracer PET. It lays the foundation for further exploration of PET-based subtyping in human cancer, thereby facilitating noninvasive molecular diagnosis.

Radiosynthesis and Analysis of (S)-4-(3-[18F]Fluoropropyl)-L-Glutamic Acid.

PURPOSE: (S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) is an L-glutamate derivative used as a PET biomarker to assess intracellular redox status in vivo through targeting of the cystine/glutamate antiporter protein, xc- transporter. In this report, we describe a radiosynthesis of [18F]FSPG for use in PET studies that address specific challenges in relation to the radiotracer purity, molar activity, and quality control testing methods. PROCEDURES: The radiosynthesis of [18F]FSPG was performed using a customised RNPlus Research automated radiosynthesis system (Synthra GmbH, Hamburg, Germany). [18F]FSPG was labelled in the 3-fluoropropylmoiety at the 4-position of the glutamic acid backbone with fluorine-18 via substitution of nucleophilic [18F]fluoride with a protected naphthylsulfonyloxy-propyl-L-glutamate derivative. Radiochemical purity of the final product was determined by radio HPLC using a new method of direct analysis using a Hypercarb C18 column. RESULTS: The average radioactivity yield of [18F]FSPG was 4.2 GBq (range, 3.4-4.8 GBq) at the end of synthesis, starting from 16 GBq of [18F]fluoride at the end of bombardment (n = 10) in a synthesis time of 50 min. The average molar activity and radioactivity volumetric concentration at the end of synthesis were 66 GBq µmol-1 (range, 48-73 GBq µmol-1) and 343-400 MBq mL-1, respectively. CONCLUSION: Stability tests using a 4.6 GBq dose with a radioactivity volumetric concentration of 369 MBq mL-1 at the end of synthesis showed no observable radiolysis 3 h after production. The formulated product is of high radiochemical purity (> 95%) and higher molar activity compared to previous methods and is safe to inject into mice up to 3 h after production.

Sensitive, non-immunogenic in vivo imaging of cancer metastases and immunotherapy response.

Non-invasive imaging of tumors expressing reporter transgenes is a popular preclinical method for studying tumor development and response to therapy in vivo due to its ability to distinguish signal from tumors over background noise. However, the utilized transgenes, such as firefly luciferase, are immunogenic and, therefore, impact results when expressed in immune-competent hosts. This represents an important limitation, given that cancer immunology and immunotherapy are currently among the most impactful areas of research and therapeutic development. Here we present a non-immunogenic preclinical tumor imaging approach. Based on the expression of murine sodium iodide symporter (mNIS), it facilitates sensitive, non-invasive detection of syngeneic tumor cells in immune-competent tumor models without additional immunogenicity arising from exogenous transgenic protein or selection marker expression. NIS-expressing tumor cells internalize the gamma-emitting [99mTc]pertechnetate ion and so can be detected by SPECT (single photon emission computed tomography). Using a mouse model of pancreatic ductal adenocarcinoma hepatic metastases in immune-competent C57BL/6 mice, we demonstrate that the technique enables the detection of very early metastatic lesions and longitudinal assessment of immunotherapy responses using precise and quantifiable whole-body SPECT/CT imaging.

Positron emission tomography imaging of the sodium iodide symporter senses real-time energy stress in vivo.

BACKGROUND: Tissue environment is critical in determining tumour metabolic vulnerability. However, in vivo drug testing is slow and waiting for tumour growth delay may not be the most appropriate endpoint for metabolic treatments. An in vivo method for measuring energy stress would rapidly determine tumour targeting in a physiologically relevant environment. The sodium-iodide symporter (NIS) is an imaging reporter gene whose protein product co-transports sodium and iodide, and positron emission tomography (PET) radiolabelled anions into the cell. Here, we show that PET imaging of NIS-mediated radiotracer uptake can rapidly visualise tumour energy stress within minutes following in vivo treatment. METHODS: We modified HEK293T human embryonic kidney cells, and A549 and H358 lung cancer cells to express transgenic NIS. Next, we subjected these cells and implanted tumours to drugs known to induce metabolic stress to observe the impact on NIS activity and energy charge. We used [18F]tetrafluoroborate positron emission tomography (PET) imaging to non-invasively image NIS activity in vivo. RESULTS: NIS activity was ablated by treating HEK293T cells in vitro, with the Na+/K+ ATPase inhibitor digoxin, confirming that radiotracer uptake was dependent on the sodium-potassium concentration gradient. NIS-mediated radiotracer uptake was significantly reduced (- 58.2%) following disruptions to ATP re-synthesis by combined glycolysis and oxidative phosphorylation inhibition in HEK293T cells and by oxidative phosphorylation inhibition (- 16.6%) in A549 cells in vitro. PET signal was significantly decreased (- 56.5%) within 90 min from the onset of treatment with IACS-010759, an oxidative phosphorylation inhibitor, in subcutaneous transgenic A549 tumours in vivo, showing that NIS could rapidly and sensitively detect energy stress non-invasively, before more widespread changes to phosphorylated AMP-activated protein kinase, phosphorylated pyruvate dehydrogenase, and GLUT1 were detectable. CONCLUSIONS: NIS acts as a rapid metabolic sensor for drugs that lead to ATP depletion. PET imaging of NIS could facilitate in vivo testing of treatments targeting energetic pathways, determine drug potency, and expedite metabolic drug development.

Imaging sialylated tumor cell glycans in vivo.

Cell surface glycans are involved in numerous physiological processes that involve cell-cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido-labeled N-acetyl-mannosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5-azidoneuraminic, or azidosialic acid. Azidosialic acid-labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far-red fluorophore or (111)In, there was a significant azido-labeled N-acetyl-mannosamine-dependent increase in tumor-to-tissue contrast, which was detected using optical imaging or single-photon-emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.

The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

Hyperpolarized Carbon-13 MRI for Early Response Assessment of Neoadjuvant Chemotherapy in Breast Cancer Patients.

Hyperpolarized 13C-MRI is an emerging tool for probing tissue metabolism by measuring 13C-label exchange between intravenously injected hyperpolarized [1-13C]pyruvate and endogenous tissue lactate. Here, we demonstrate that hyperpolarized 13C-MRI can be used to detect early response to neoadjuvant therapy in breast cancer. Seven patients underwent multiparametric 1H-MRI and hyperpolarized 13C-MRI before and 7-11 days after commencing treatment. An increase in the lactate-to-pyruvate ratio of approximately 20% identified three patients who, following 5-6 cycles of treatment, showed pathological complete response. This ratio correlated with gene expression of the pyruvate transporter MCT1 and lactate dehydrogenase A (LDHA), the enzyme catalyzing label exchange between pyruvate and lactate. Analysis of approximately 2,000 breast tumors showed that overexpression of LDHA and the hypoxia marker CAIX was associated with reduced relapse-free and overall survival. Hyperpolarized 13C-MRI represents a promising method for monitoring very early treatment response in breast cancer and has demonstrated prognostic potential. SIGNIFICANCE: Hyperpolarized carbon-13 MRI allows response assessment in patients with breast cancer after 7-11 days of neoadjuvant chemotherapy and outperformed state-of-the-art and research quantitative proton MRI techniques.

Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER+ Breast Cancer.

PIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance.

Imaging Cancer Metabolism with Positron Emission Tomography (PET).

Positron emission tomography (PET) enables the noninvasive spatiotemporal analysis of cancer metabolism in vivo. Both natural and nonnatural PET tracers have been developed to assess metabolic pathways during tumorigenesis, cancer progression, and metastasis. Here we describe the dynamic in vivo PET/CT imaging of the glucose analogue [18F]fluoro-2-deoxy-D-glucose (FDG), taking into consideration the methodology for alternative metabolic PET substrates.

Magnetic Resonance Imaging Is More Sensitive Than PET for Detecting Treatment-Induced Cell Death-Dependent Changes in Glycolysis.

Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death.

Assessment of Tumor Redox Status through (S)-4-(3-[18F]fluoropropyl)-L-Glutamic Acid PET Imaging of System xc - Activity.

The cell's endogenous antioxidant system is vital to maintenance of redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc - maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here, we show that tumor cell retention of a system xc --specific PET radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment. SIGNIFICANCE: [18F]FSPG PET imaging provides a sensitive noninvasive measure of tumor redox status and provides an early marker of tumor response to therapy.See related commentary by Lee et al., p. 701.

[18F]fluoroethyltyrosine-induced Cerenkov Luminescence Improves Image-Guided Surgical Resection of Glioma.

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O­(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA). Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining. Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection. Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.

Brain Tumor Imaging.

Modern imaging techniques, particularly functional imaging techniques that interrogate some specific aspect of underlying tumor biology, have enormous potential in neuro-oncology for disease detection, grading, and tumor delineation to guide biopsy and resection; monitoring treatment response; and targeting radiotherapy. This brief review considers the role of magnetic resonance imaging and spectroscopy, and positron emission tomography in these areas and discusses the factors that limit translation of new techniques to the clinic, in particular, the cost and difficulties associated with validation in multicenter clinical trials.

Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and 99mTc- and 111In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation (R > 0.9, P < 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99mTc-C2Am and 111In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99mTc-C2Am and 111In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99mTc- and 111In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation.

Imaging tumor metabolism using positron emission tomography.

Positron emission tomography (PET) is an extraordinarily sensitive clinical imaging modality for interrogating tumor metabolism. Radiolabeled PET substrates can be traced at subphysiological concentrations, allowing noninvasive imaging of metabolism and intratumoral heterogeneity in systems ranging from advanced cancer models to patients in the clinic. There are a wide range of novel and more established PET radiotracers, which can be used to investigate various aspects of the tumor, including carbohydrate, amino acid, and fatty acid metabolism. In this review, we briefly discuss the more established metabolic tracers and describe recent work on the development of new tracers. Some of the unanswered questions in tumor metabolism are considered alongside new technical developments, such as combined PET/magnetic resonance imaging scanners, which could provide new imaging solutions to some of the outstanding diagnostic challenges facing modern cancer medicine.